Combination of unsaponifiable lipids combined with polyphenols and/or catechins for the protection, treatment and repair of cartilage in joints of humans and animals

ABSTRACT

The present invention relates to a composition for the protection, treatment and repair of cartilage in humans and animal joints. The composition contains a combination of unsaponifiable lipids together with one or more of polyphenols and/or catechins. The composition or kit may contain avocado:soybean unsaponifiables (ASU) and green tea.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/296,031, filed Jun. 4, 2014, now U.S. Pat. No.9,572,791, which is a continuation application of U.S. patentapplication Ser. No. 12/780,642, filed May 14, 2010, now U.S. Pat. No.8,808,770, which is a divisional application of U.S. patent applicationSer. No. 11/889,060, filed Aug. 8, 2007, which is a continuationapplication of U.S. patent application Ser. No. 11/192,362, filed Jul.29, 2005, which claims the benefit of U.S. Provisional PatentApplication No. 60/592,322, filed on Jul. 30, 2004, the contents of allof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a composition for the protection,treatment and repair of cartilage in humans and animal joints.

BACKGROUND OF THE INVENTION

Degenerative joint disease (DJD) results from the cumulative effects ofan imbalance between synthesis and degradation of components ofcartilage extracellular matrix. This imbalance is associated withdisregulated activity of inflammatory cytokines and production ofprostaglandins that mediate pain. Progression of the disorder occurswhen this imbalance persists. Agents that act to increase synthesis ofcartilage components and suppress the activity of specific cytokines,particularly of interleukin-1-beta (IL-1-β) and tumor necrosis factoralpha (TNF-α), have the potential to relieve symptoms of DJD and stopits progression. Both of these cytokines act through mechanisms thatinvolve generation of reactive oxygen species (ROS) and reactivenitrogen species (RNS) that lead to suppression of synthesis ofcartilage matrix proteoglycans along with increased production ofprostaglandins, nitric oxide, and activation of metalloproteinases thatdegrade cartilage proteins. A combination of agents that is capable ofinteracting with these mechanisms in different ways and to stimulate theanabolic response needed in chondrocytes for maintenance and/orrestoration of matrix components could be effective in the protection,treatment and repair of connective tissue.

Polyphenols and Catechins

Various polyphenols can be sourced from berries and other fruits.Catechins are found primarily in teas. The preferred source ofpolyphenols and catechins are sourced from green tea. Green tea containsa mixture of catechins, including epicatechin (EC), epigallocatechin(EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG).These catechins have potent antioxidant activity, acting as scavengersof the free radicals (ROS and RNS) involved in damage to cells. Theyalso act by chelating metals that catalyze production of ROS (1). Thisantioxidant activity may interfere with the damaging effects of agents,e.g. fibronectin fragments (Fn-f) and cytokines, that can cause DJD.Antioxidants block the effects of Fn-f, which include increasedexpression and activity of both cytokines IL-1 and TNF-α (2,3). Inaddition, recent studies have shown that green tea polyphenolssignificantly reduce the incidence of collagen-induced arthritis in micethat was associated with reduced expression of TNF-α and cyclooxygenase2, a TNF-α regulated enzyme that catalyzes the production ofprostaglandin E2 (4). Other studies have shown that the EGCG in greentea inhibits IL-1 induced expression of nitric oxide synthase and nitricoxide production and suppresses activation of nuclear factor-kB, a keystep in initiation of the cytokine effects (5). Furthermore, thecatechins in green tea were recently shown to potently inhibitaggrecanase activities known to be involved in the early stages ofdestruction of cartilage proteoglycans (6). Thus, the components ofgreen tea have the potential to ameliorate the cause and the symptoms ofDJD through multiple mechanisms. The green tea may be administered as anextract or standardized to polyphenols or catechins.

Unsaponifiable Lipids

Vegetable oils, including coconut, peanut and safflower oil to name afew, are possible components of the human diet that contain an importantclass of compounds termed unsaponifiable lipids. One rich source ofunsaponifiable lipids are avocados and soybean oil, with avocado havingapproximately four times more lipids than those found in other commonlyeaten fruits. Many of the health benefits of soy, widely used as astaple food in Asian countries, may be due to this unsaponifiablefraction which also includes isoflavones. Unsaponifiable lipids aredefined as the material which does not react with basic agents to formsoaps. Included in this class are phytosterols, fat soluble vitamins A,D, E and K, bioflavinoids, phytoestrogens and small organic moleculescalled terpenes. The major components by weight of the unsaponifiablelipid fraction are a group of compounds called phytosterols which differslightly in structure and include beta sitosterol, campesterol andstigmasterol. Of the many beneficial actions attributed to phytosterols,their ability to reduce pain, swelling, and tissue injury in joints isproposed. With aging and stress, increased amounts of phytosterols arethought to be necessary to maintain normal biological functions (1,2).

Biological Activity

Phytosterols have potent biological activities, some of which reduce thejoint pain and swelling characteristic of inflammatory joint disordersas well as symptoms characteristic of trauma or aging related jointdisorders. In addition, these agents beneficially alter the bodiesimmune response in a fashion which helps to minimize joint tissuedestruction attributed to an allergic tissue reaction. Studies indicatephytosterols are active in immune modulation (3) and haveanti-inflammatory and antipyretic activity (4). Most of these activitiesare a result of the effect of phytosterols inhibiting the production byinflammatory cells of chemical agents (cytokines) which cause tissuedamage and stimulation of anabolic activity of chondrocytes.Avocado/soybean oil unsaponifiables (ASU) is a preferred source ofunsaponifiable lipids for the protection, treatment and repair ofconnective tissue conditions.

In Vitro Tests

Prior researchers have conducted in vitro tests on ASU. The in vitromodel previously used to test ASU is based on monitoring reduction ofinterleukin-1 (IL-1)-induced metalloprotease activity, nitric oxide orprostaglandin synthesis (all agents associated with cartilagedegradation, tissue inflammation, or pain) (5-9). ASU was shown toinhibit the action of IL-1 at doses of 1 to 10 ug/ml. A combination of2:1 soy:avocado was found to be even more effective than either agentalone (5,8). It was also noticed that this mixture was the maincombination that was effective in decreasing production ofcollagenolytic activity (9). Fibroblasts also appear to be responsive toASU. Metalloprotease activity (MMP-2 and MMP-3) was inhibited at lowdoses of ASU and at higher doses the tissue-inhibitors ofmetalloproteases was increased (7). The above mentioned assays could berelated to the beneficial physiological (symptomatic relief) effects.Other indices tested relate to a possible increase capacity for repairand regeneration of articular cartilage. For example, the anticatabolicactivity of ASU was shown to be paired with a direct effect of ASU onstimulating collagen and proteoglycan production, possibly by increasingtransforming growth factor-beta 1 and 2 synthesis (6). Research in ourlaboratory also has shown that ASU is effective in improving cartilagesynthesis in-vitro.

In Vivo Tests in Animals

Prior researchers have conducted in vivo animal tests on ASU. Anincrease in collagen synthesis was observed in a carrageenan inducedgranuloma in the “Hairless” rat following 15 days of percutaneouslyapplied ASU (10). Mice that had subcutaneous implantation of ratarticular cartilage wrapped in cotton were treated orally for 2 weekswith the unsaponifiable fraction of either ASU, avocado alone or soybeanalone daily for 2 weeks (13 mg/kg avocado or 26 mg/kg soya or both in aratio of 1:2 at a dose of 39 mg/kg). Parameters measured includedproteoglycans and hydroxyproline content of the cartilage. Resultsindicate that unsaponifiables of both avocado and soybean reduced thedegeneration of proteoglycans and hydroxyproline content of theimplanted cartilage that was induced by the granuloma tissue. A greatereffect was seen using a combination of avocado and soybean, an effectwhich was dose dependent (11).

A third more pertinent study involved oral administration of ASU (900mg/weekday) to meniscectomized sheep for 3 and 6 months (12). In thismodel a “subtle but statistically significant protective effect onarticular cartilage” was noted from computerized image analysis ofhistological stained cartilage.

Many plants contain similar type agents generally classified asunsaponifiable lipids. Potent anti-inflammatory activity has also beenassociated with these sources. For example, Park et al (13) foundanti-inflammatory activity in an ethanolic extract of cactus which wasidentified as beta sitosterol. Using the carrageenan paw oedema model inrats, beta sitosterol was found to be one of the potentanti-inflammatory agents in extracts of Verbena officinalis (14). Theisolated sterols stigmasterol and beta sitosterol were potentanti-inflammatory agents when administered topically and in a chronicinflammation model in the mouse (15). A sterol fraction containing 7.6%campesterol, 28.4% stigmasterol and 61.1% beta sitosterol demonstratedanti-inflammatory activity in the carrageenan paw oedema model in miceafter oral administration of 30 and 60 mg/kg (16).

In Vivo Tests in Humans

Prior researchers have conducted in vivo tests in humans on ASU.Individuals with primary femorotibial or hip OA of at least six monthsduration were dosed with 300 mg of a 2:1 avocado:soybean unsaponifiablepreparation for 3 months. Indices measured included NSAID intake,Lequesne's index and physician visual analog scale for pain andfunctional index. All indices showed improvement at p<0.01 or better(17). In a double-blind study, individuals with knee OA (femoro-tibial)were dosed with 300 or 600 mg of an avocado:soybean unsaponifiable forthree months. Indices measured included NSAID and analgesic intakebetween day 30 and day 90. All indices improved with treatment at p<0.01or better with NSAID intake decreasing by more than 50% in 71% of theindividuals compared to 36% in individuals receiving placebo. Lequesne'sindex dropped by 3.9 and 2.9 points in ASU 300 and 600 mg respectivelyagainst 1.6 in placebo. Similar results were observed in individualsgiven 300 or 600 mg (18). Eighty-five out of 164 individuals withpainful primary OA of the knee or hip were dosed for 6 months with 300mg unsaponifiables after a 15 day washout period for NSAIDs. Efficacywas determined by Lequesne's functional index, visual analog pain scale,intake of NSAIDs and overall disability score. Pain decreased from ascore of 56.1 to 35.3 in the ASU group and from 56.1 to 45.7 in theplacebo group (p<0.003). Values for NSAID intake showed consumptionlower in ASU group (48% versus 63%). Lequesne's test score decreasedfrom 9.7 to 6.8 in the ASU group and from 9.4 to 8.9 in the placebogroup. The overall success rate was 39% in the ASU group and 18% in theplacebo group. Improvement was more marked in individuals with hip OAand a residual effect was observed at month eight (19).

One hundred sixty three individuals in this pilot 2 year studyevaluating structural changes in the hip joint. Individuals with painfulprimary OA of hip and joint space > or = to 1 mm. (Kellgren grade 1 to3) with at least a 6 month history of pain and AFI index > or = to 4.Primary assessment was decrease in joint space width performed instanding, position. Results indicate a failure to demonstratesignificant reduction in progression of joint space loss compared toplacebo. ASU did reduce progression of joint space loss (20).

Rationale

We believe that polyphenols and catechins, especially from green tea,together with unsaponifiable lipids, especially from ASU, can havebeneficial effects on the biochemical processes that underliedevelopment and/or progression of DJD. In our invention, by “green tea”we mean green tea available as a tea, as well as green tea available asan extract, such as in a powder form. The anti-oxidant activity ofcomponents of green tea have the capacity to attack the free radicalsknown to be involved in disregulated cytokine activity inosteoarthritis. The additional capacity to inhibit specific proteasesinvolved in cartilage degradation provides an additional mechanism forcombating DJD. The anti-inflammatory activity of avocado and soybeanunsaponifiables can also attack cytokine signaling, but apparently byless understood mechanisms. They also provide direct anabolic activitythat is needed to restore the balance between anabolic and catabolicactivity. A combination of unsaponifiable lipids with one or more ofpolyphenols or catechins should work well together. Thus the presentinvention consists of a combination of these agents over a range ofdoses.

Dosage Ranges and Preferred Sources

Polyphenols/catechins: about 3 mg to about 10 grams, with the preferredsource of polyphenols being green tea. The green tea may be combinedwith phosphatidylcholine to improve absorption.

Unsaponifiable lipids: about 5 mg to about 12 grams, with the preferredsource being ASU.

EXAMPLES

The following examples are merely illustrative of the present inventionand are not to be considered as limiting the invention, which isproperly delineated in the following claims. Moreover, it should benoted that the use of the present or future tense in these examples isreflective of the fact that the examples are prophetic. MPEP ¶608.01(p).

Example 1

A 70 kg 60 year old man has knee joint pain and radiologically diagnoseddegenerative joint disorder. Intake on a daily basis of a supplementthat contains 100 mg green tea extract and 300 mg avocado-soybeanunsaponifiables reduces these symptoms.

Example 2

A 20 kg 6 year old Labrador retriever has degenerative joint disorderlocalized to the hip and has difficulty rising and ascending stairs.Intake on a daily basis of a supplement containing 20 mg green teaextract and 30 mg avocado-soybean unsaponifiables reduces thesesymptoms.

Example 3

A 5 kg cat is diagnosed with spinal arthritis and cannot jump on thewindowsill. The cat is administered 3 mg of polyphenols and catechinsand 5 mg avocado soybean unsaponifiables. The cat improves dramaticallyand is now capable of jumping on the windowsill.

Example 4

A 1000 kg horse is diagnosed with bone spavin and treated with 3 gramsof epigallocatechin gallate and 4 grams of ASU. The horse respondsdramatically.

REFERENCES FOR GREEN TEA

-   1. Higdon, J. V. and Frei B., Tea Catechins and Polyphenols: Health    Effects, Metabolism, and Antioxidant Functions. Critical Reviews in    Food Science and Nutrition 4389-143, 2003.-   2. Homandberg, G. A. and Wen F. H-Fibronectin Fragment Mediated    Cartilage Chondrolysis. J. Suppression by Anti-oxidants. Biochimica    Biophysica Acta 1317:134-142, 1996.-   3. Homandberg, G. A., Hui F., Wen C., Purple C., Bewsey K., Koepp    H., Huch K. and Harris A. Fibronectin-Fragment-Induced Cartilage    Chondrolysis is Associated with Release of Catabolic Cytokines.    Biochemical Journal 321:751-757, 1997.-   4. Haqqi T, Anthony D. D., Gupta S., Ahmad N., Lee M. S.,    Kumar G. K. and Mukhtar H. Prevention of Collagen-induced Arthritis    in Mice by a Polyphenolic Fraction From Green Tea. Proceedings    National Academy Science USA 96:4524-4529, 1999.-   5. Singh R., Ahmed S., Islam N., Goldberg V. M. and Haqqi T. M.    Epigallocatechin-3-Gallate Inhibits Interleukin-1B-Induced    Expression of Nitric Oxide Synthase and Production of Nitric Oxide    in Human Chondrocytes. Arthritis & Rheumatism 45:2079-2086, 2002.-   6. Vankemmelbeke M. N., Jones, G. C., Fowles C., Ilic M. Z.,    Handley C. J., Day A. T., Knight C. G., Mort J. S. and Buttle,    D., J. Selective inhibition of ADAMTS-1, -4, and -5 by Catechin    Gallate Esters. European Journal Biochemistry 270: 2394-2403, 203.

REFERENCES FOR ASU

The following references regarding ASU are herein incorporated byreference in their entirety into this specification.

-   1. Ling W. H., Jones P. J. H.: Minireview. Dietary Phytosterols: A    Review of Metabolism, Benefits and Side Effects. Life Sciences 57:    195-206, 1995.-   2. DeJong A., Plat J., Mensink R. P.: Metabolic Effects of Plant    Sterols and Stanols (Review). J Nutri Biochem 14: 362-369, 2003.-   3. Bouic P. J.: The Role of Phytosterols and Phytosterolins in    Immune Modulation: A Review of the Past 10 Years. Curr Opin Clin    Nutr Metab Care 4: 471-475, 2001.-   4. Gupta M. B., Nath R., Srivastava N., Shanker K., Kishor K.,    Bhargava K. P.: Antiinflammatory and Antipyretic Activities of Beta    Sitosterol. Planta Med 39: 157-163, 1980.-   5. Henrotin Y. E., Labasse A. H., Jaspar J. M., DeGroote D. D.,    Zheng S. X., Guillou G. B., Reginster J. Y.: Effects of Three    Avocado/Soybean Unsaponifiable Mixtures on Metalloproteinasses,    Cytokines and PGE2 Production by Human Articular Chondrocytes. Clin    Rheumatol 17: 31-39, 1998.-   6. Boumediene K., Felisaz N., Bogdanowiez P., Galera P., Guillou G.    B., Pujol J. P.: Avocado/Soya Unsaponifiabies Enhance the Expression    of Transforming Growth Factor Beta I and Beta 2 in Cultured    Articular Chondrocyctes. Arthritis Rheum 42: 148-156, 1999.-   7. Kut-Lasserre C., Miller C. C., Ejeil A. L., Gogly B., Dridi M.,    Piccardi N., Guillou B., Pellat B., and Godeau. Effect of Avocado    and Soybean Unsaponifiables on Gelatinase A (MMP-2), Stromelysin 1    (MMP-3) and Tissue Inhibitors of Matrix Metalloproteinase (TMP-1 and    TIMP-2) Secretion by Human Fibroblasts in Culture. J Periodontol 72:    1685-1694, 2001.-   8. Henrotin Y. E., Sanchez C., Deberg M. A., et al: Avocado/Soybean    Unsaponifiables Increase Aggrecan Synthesis and Reduce Catabolic and    Proinflammatory Mediator Production by Human Osteoarthritic    Chondrocytes. J Rheumatol 30: 1825-1834, 2003.-   9. Mauviel A., Loyau G., Pujol J. P.: Effect of Unsaponifiable    Extracts of Avocado and Soybean (Piascledine) on the Collagenolytic    Action of Cultures of Human Rheumatoid Synoviocytes and Rabbit    Articular Chondrocytes Treated with Interleukin-1. Rev Rhurn Mal    Osteoartic 58: 241-245, 1991.-   10. Lamaud M. E., Miskulin M., Robert A. M., Wepierre J.:    Biochemical Modifications of Connective Tissue Induced by the    Non-Saponifiables of Avocado and Soyabean Oils Administered    Percutaneously in the Hairless Rat. Pathol Biol 26: 269-274, 1978.-   11. Khayyal M. T., El-Ghazaly M. A.: The Possible    “Chondroprotective” Effect of the Unsaponifiable Constituents of    Avocado and Soya In Vivo. Drugs Exptl Clin Res 24: 41-50, 1998.-   12. Cake M. A., Read R. A., Guillou B., Ghosh P.: Modification of    Articular Cartilage and Subchondral Bone Pathology in an Ovine    Meniscectomy Model of Osteoarthritis by Avocado and Soya    Unsaponifiables (ASU). Osteoarthritis & Cartilage 8: 404-411, 2000.-   13. Park E. H., Kahng J. H., Lee S. H., Shin K. H.: An    Antiinflammatory Principle from Cactus. Fitoterapia 72: 288-290,    2001.-   14. Deepak M., Handa S. S.: Antiinflammatory Activity and Chemical    Composition of Extracts of Verbena Officinalis. Phytother Res 14:    463-465, 2000.-   15. Gomez M. A., Saenz M. T., Garcia M. D., Fernandez M. A.: Study    of the Topical Antiinflammatory Activity of Achillea Ageratum on    Chronic and Acute Inflammation Models Z Naturforsch [C] 54:937-941,    1999.-   16. Navarro A., DeLasHeras B., Villar A.: Antiinflammatory and    Immunomodulating Properties of a Sterol Fraction from Sideritis    Foetens Chem. Biol Pharm Bull 24: 470-473, 2001.-   17. Blotman F., Maheu E., Wuiwik A., Caspard H., Lopez A.: Efficacy    and Safety of Avocado/Soybean Unsaponifiables in the Treatment of    Symptomatic Osteoarthritis of the Knee and Hip. A Prospective,    Multicenter, Three-Month Randomized, Double-Blind,    Placebo-Controlled Trial. Rev Rhum Engl Ed 64: 825-834, 1997.-   18. Appelboom T., Schuermans J., Verbuggen G., Henrotin Y.,    Reginster J. Y.: Symptoms Modifying Effect of Avocado/Soybean    Unsaponifiables (ASU) in Knee Osteoarthritis. Scand J Rheumatol 30:    242-247, 2001.-   19. Maheu E., Mazieres B., Valat J. P., Loyau G., LeLoet X.,    Bourgeois P., Grouin J. M, Rozenberg S.: Symptomatic Efficacy of    Avocado/Soybean Unsaponifiables in the Treatment of Osteoarthritis    of the Knee and Hip. Arthritis & Rheum 41:81-91, 1998.-   20. Lequesne M., Maheu E., Cadet C., Dreiser R. L.: Structural    Effect of Avocado/Soybean Unsaponifiables on Joint Space Loss in    Osteoarthritis of the Hip. Arthritis & Rheum 47:50-58, 2002.

What is claimed:
 1. A formulation for oral administration for thetreatment and repair of connective tissue and reduction of inflammationin joints of a human or animal in need thereof comprisingavocado/soybean unsaponifiables (ASU) and epigallocatechin gallate(EGCG) provided in synergistically effective amounts.
 2. The formulationof claim 1, wherein the ASU is in an amount of about 5 mg to about 12 g.3. The formulation of claim 1, wherein the EGCG is in an amount ofbetween about 3 mg to about 10 g.
 4. The formulation of claim 1, whereinthe EGCG is sourced from green tea or green tea extract.
 5. Theformulation of claim 4, wherein the formulation provides a dose of about0.04 mg/kg to about 40 mg/kg of green tea extract to the human oranimal.
 6. The formulation of claim 1, wherein the formulation providesa dose of about 0.04 mg/kg to about 4 mg/kg of EGCG to the human oranimal.
 7. The formulation of claim 1, wherein the formulation providesa dose of about 1 mg/kg to about 5 mg/kg of ASU to the human or animal.8. The formulation of claim 1, further comprising phosphatidylcholine.